Lettuce (Lactuca sativa L.) seeds are inhibited from germinating above an upper temperature limit that is dependent upon cultivar, growing conditions and seed treatments. Thermoinhibition is accompanied by an increasing sensitivity of germination to reduced water potentials (ψ). We have employed a water relations analysis (hydrotime model) of seed germination rates to investigate the basis of thermoinhibition. Germination rates can be characterized by the distribution of base water potentials among seeds in the population (ψb(g)) and a hydrotime constant indicating the total accumulated hydrotime (MPa · h) above the base required for radicle emergence. The hydrotime model adequately described germination time courses across a range of ψ at both high and low temperatures. Increasing temperature caused the ψb(g) distributions to become more positive, accounting for the greater sensitivity to ψ and inhibition of germination. Increases in embryo osmotic potential and in the turgor yield thresholds of both the radicle and the endosperm/pericarp envelope contributed to this change. Seed priming (prehydration and drying) treatments speeded germination by reducing the hydrotime requirement. Priming also resulted in smaller increases in ψb(g) at high temperature, alleviating thermoinhibition by lowering the embryo yield threshold sufficiently to compensate for the increased endosperm resistance. The beneficial effects of priming in lettuce appear to occur primarily in the embryo, rather than in the surrounding envelope tissues.

Excised Helianthus annuus L. embryos became dormant during the third week after anthesis. At this stage a short drying treatment (3 d) led to a slight improvement in germination but a 6-week dry storage caused a complete release from dormancy. The short drying treatment, however, elicited the embryos' response to an exogenous concentration of GAs which was unable to promote germination of fresh embryos. It therefore appeared that a short drying treatment changed the sensitivity to GAs but was not capable of directing embryo metabolism completely towards a germinative mode. Moreover, this drying treatment reduced considerably the ABA content in both the axis and the embryo. Nevertheless, no correlation could be established between germinability and the ABA content since the amount of ABA was not modified by the 6-week dry storage. The key step for predisposing the seeds to germinate is the suppression of the capacity for ABA synthesis in the axis, a suppression which takes place during 6-week dry storage.

In view of the finding that dry seeds may exist in the glassy state, a brief review of the commonest methods of detection and quantification of the glass transition is presented. While the glassy state may contribute to seed tolerance of desiccation, it does not appear to account for desiccation tolerance. Its major function in dry seeds may be its contribution to the stability of the seed components during storage.

Seed germination rates are sensitive to both temperature (T) and water potential (ψ). The times to germination of seeds imbibed at suboptimal T and/or reduced ψ are inversely proportional to the amounts by which T exceeds a base temperature (Tb) and ψ exceeds a base water potential (ψb). Germination rates across a range of suboptimal T and ψ can be normalized on the basis of the hydrothermal time accumulated in excess of these thresholds. However, seeds can also progress metabolically toward germination even at T or ψ too low to allow radicle emergence to occur. Seeds preimbibed at low ψ and dried back, or primed, germinate more rapidly upon subsequent reimbibition. We show here that the increase in germination rates of tomato (Lycopersicon esculentum Mill.) seeds resulting from seed priming is linearly related to the hydrothermal time accumulated during the priming treatment. The threshold temperature (Tmin = 7.05°C) and water potential (ψmin = −2.50 MPa) for metabolic advancement were considerably lower than the corresponding thresholds for radicle emergence of the same seed lot (Tb = 11°C; ψb = −0.71 MPa), allowing the accumulation of hydrothermal priming time that is subsequently expressed as more rapid germination when T or ψ increase. The hydrothermal time model can now be applied to quantify and analyse germination rates of seeds across the entire range of suboptimal T and ψ at which metabolic progress toward radicle emergence is possible.

A computer-aided comparison of tomato (Lycopersicon esculentum Mill.) seed protein patterns, obtained after two-dimensional gel electrophoresis, was made for three different extraction procedures: TCA acetone/lysis buffer, lysis buffer only and modified Laemmli/lysis buffer. Comparison of the isolation methods showed that about half of the amount of proteins detected was common in each method. Also, proteins specific to some isolation methods were detected. Protein synthesis during imbibition was monitored using 35S-methionine. After labelling the proteins were extracted using TCA acetone/lysis buffer. Following two-dimensional gel electrophoresis the gels were first silver stained, to give a general picture of all proteins present in the seed, then the gels were exposed to a film for autoradiography. Comparison of the in vivo-synthesized protein patterns and the silver-stained proteins revealed that from day 0 to day 1 the protein pattern was changed but the total number of different spots was similar. After 1 day of imbibition, the number of protein spots increased greatly and the protein pattern changed again.

A water relations (hydrotime) model was employed to characterize the responses of lettuce (Lactuca sativa L.) seed germination to temperature, water potential (ψ), endosperm disruption, and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). The base water potentials (ψb), standard deviations of base water potential and hydrotime constants (θH) estimated by the model were used to predict the germination rates at reduced ψ and supraoptimal temperatures in the presence or absence of ACC. The distribution of ψb values among seeds in the population (ψb(g)) increased (became more positive) as the imbibition temperature increased, indicating a greater sensitivity of seeds to reduced ψ at thermoinhibitory temperatures. Slitting the seeds at the cotyledonary end to disrupt the integrity of the endosperm extended the high temperature range for germination and maintained lower ψb(g) distributions compared with intact seeds as temperature increased. ACC (10 mm) in the imbibition solution raised the upper temperature limit for germination by about 2°C, and also lowered ψb(g) values by 0.1 to 0.2 MPa. The effect of ACC on ψb(g) was more pronounced at higher imbibition temperatures and in slit seeds. ACC (via conversion to ethylene) extends the high temperature limit for lettuce seed germination by acting in the embryo to maintain a lower ψ threshold for the initiation of growth as temperature increases. Evidence from other species as well suggests that this may be a general mechanism for the action of ethylene in promoting seed germination.

The development of techniques for somatic embryogenesis in conifers has led to rapid advances in the ability to culture conifer tissue in vitro. Somatic embryogenesis now provides a means to clonally propagate commercially valuable conifers of several species and offers an in vitro experimental system for studying embryogenesis. As a result of these developments, the conifer zygotic and somatic embryo system has recently attracted the attention not only of the cell biologist but also of biochemists and molecular biologists. In this review I have attempted to consolidate the recent information on ultrastructure, biochemical and molecular aspects of conifer zygotic and somatic embryogenesis. In the first section, salient features of zygotic embryogenesis are highlighted followed by biochemical and ultrastructural characterization of development with reference to storage reserve deposition. In the second section, detailed characterization of seed storage proteins, changes in gene expression with emphasis on storage protein genes and Lea genes during zygotic and somatic embryogenesis are described. Effect of culture treatments such as abscisic acid, osmoticum and desiccation on storage protein gene expression in somatic embryos is discussed. Based on these studies, comparisons are presented between angiosperm and gymnosperm embryogeny regarding the unique morphological and developmental patterns of conifer embryogenesis. Finally, a brief discussion of recent progress in biochemical and molecular characterization of conifer seed germination is presented.

Both temperature (T) and water potential (ψ) have consistent and quantifiable effects on the rate and extent of seed germination (radicle emergence). Germination at suboptimal T can be characterized on the basis of thermal time, or the T in excess of a base (Tb) multiplied by the time to a given percentage germination (tg). Similarly, germination at reduced ψ can be characterized on a hydrotime basis, or the ψ in excess of a base (ψb) multiplied by tg. Within a seed population, the variation in thermal times to germination for a specific percentage (g) is based upon the normal distribution of ψb values among seeds (ψb(g)). Germination responses across a range of suboptimal T and ψ might be accounted for by a general hydrothermal time model incorporating both T and ψ components. We tested this hypothesis for tomato (Lycopersicon esculentum Mill.) seeds of two genotypes differing in germination rates and tolerance of suboptimal T and ψ. For combinations of T (10−25°C) and ψ (0 to −0.9 MPa), a general hydrothermal time model accounted for approximately 75% of the variation in times to germination within the seed populations of both genotypes, and over 96% of the variation in median germination rates. However, ψb(g) distributions were sensitive to both the T and ψ of imbibition, resulting in a poor fit of the model to specific time course data. Analysis of germination timing separately for low and high ψ ranges within a given T resulted in specific models accounting for 88−99% of the variation in individual germination times and >99% of the variation in madian germination rates. Thus, for a given T and ψ range, the hydrotime model closely matched tomato seed germination time courses. Accumulated hydrothermal time accounted well for germination rates at ψ> −0.5 MPa across suboptimal T if ψb(g) was allowed to vary with T. Germination did not show a consistent response to T at ψ < −0.5 MPa, and estimated Tb values varied over different T ranges. Generalization of the hydrothermal time model across the entire range of suboptimal T and ψ was limited by physiological adjustments of the seeds to their current environment. The hydrothermal time model detected and quantified these adjustment processes that would otherwise not be evident from inspection of germination time courses. Temperature and water potential influence the time to germination via physiological mechanisms that reciprocally interact.

Seeds from an assortment of 28 grass species (including two cereals), chiefly representing different tribes of the sub-family Pooideae were tested for the presence of BASI-like proteins using Western blotting techniques and antisera raised against the barley amylase/subtilisin inhibitor. A single protein species of the same molecular weight as the BASI protein was detected in each member of the tribes Triticeae and Bromeae tested. Members of the Triticeae and Bromeae were the only species to produce high pi α-amylase isoenzymes following germination of the grain in addition to low pl forms which were present in all species tested. Anti-BASI antibody binding patterns for the other grasses examined were variable with the strongest staining being observed for Lolium species. In these species, either a double or single protein band fractionally larger than the BASI protein was recognized. In most other cases antibody binding was barely or not detectable. Inhibitor preparations from Hordeum vulgare and Lolium perenne were effective at inhibiting wheat α-amylase but neither had any effect against enzyme produced by germinating L. perenne.

The effect of temperature on the Pfr level required to induce germination in lettuce cv. Grand Rapids was investigated. Various Pfr levels were established in seeds, by irradiation with different fluences of red light, or by saturating irradiations with narrow bandwidth light. Both approaches indicated that increasing Pfr levels caused a progressive increase in the upper temperature limit for germination. The Pfr level required to induce 50% germination after a single light pulse increased from about 11% at 15° and 20°C to 86% at 30.5°C. Repeated red light irradiations raised the GT50 (temperature giving 50% germination) by about 2°C, suggesting that Pfr reversion limited germination at high temperatures. However, a large increase in Pfr level required to induce 50% germination was still apparent at high temperatures, even when relatively constant levels of Pfr were maintained by repeated irradiations with narrow bandwidth light. These results indicate that following a single light pulse, germination at high temperatures is limited solely by the availability of Pfr. The precise position of the upper temperature limit for germination is influenced by reversion; however, the underlying cause of thermoinhibition relates to an increasing requirement for Pfr at elevated temperatures.

Stachyose, raffinose and related flatulence-producing oligosaccharides (α-galactosyl derivatives of sucrose) are associated with desiccation tolerance and storability of seed germplasm. Orthodox seeds of species with a sucrose-to-oligosaccharide ratio of <1.0 have storability half-viability periods >10 years while those >1.0 have a storability half-viability period <10 years. Seeds vary in their composition of oligosaccharides and some accumulate α-galactosyl derivatives of cyclitols. Known and proposed pathways for biosynthesis of soluble oligosaccharides, cyclitols and galactosyl derivatives of cyclitols are presented. Axes, cotyledons, embryos or seeds of 19 species in 7 families (all orthodox seeds) were analysed for sucrose, galactosyl derivatives of sucrose, cyclitols and galactosyl derivatives of cyclitols by high resolution gas chromatography. Sucrose and myo-inositol are universally present and galactinol is present in seeds accumulating stachyose series oligosaccharides. Seeds of some species of Leguminosae accumulate mostly stachyose series oligosaccharides, whereas seeds of other species accumulate varying levels of galactosyl derivatives of cyclitols in addition. Castor bean (Euphorbiaceae) seeds accumulate galactinol and buckwheat (Polygonaceae) embryos accumulate galacto-chiro-inositol instead of the stachyose series oligosaccharides. The mass ratio of sucrose:non-sucrose is related to storability and is applicable to seeds accumulating cyclitol derivatives. Galactinol and galacto-chiro-inositol are proposed to function in the same role as raffinose and stachyose in facilitating desiccation tolerance and storability.

Wheat seed lots of different viability were obtained by an accelerated ageing technique in which seeds were hydrated up to 12.5–14.5% on a fresh weight basis and stored at 35°C. Ageing was accompanied by a progressive delay of the onset of germination and a decrease of [35S]-methionine uptake and incorporation into soluble proteins of the imbibing embryos. A detailed analysis of newly synthesized proteins by two-dimensional polyacrylamide gel electrophoresis showed that normal protein patterns during the germination process were impaired by ageing. Only two groups of polypeptides with molecular masses of 84.9–69.5 and 66.6–60.2 kDa, respectively, were actively produced while most proteins decreased or were definitely degraded. Our results suggest that the biochemical changes required for germination are gradually opposed by a deterioration process and that some newly-synthesized polypeptides can be taken as markers in assessing seed survival of ageing stress.

A study was conducted to assess the effect of the supply of phosphorus and the major cations on the mineral nutrient composition of whole mature wheat grains and the electron-dense globoid crystals found in the aleurone cells. Ears of wheat plants, growing in the field, were detached after anthesis. The ears were cultured to maturity in a sterile control solution which contained all essential elements. Other ears were cultured in solutions which contained no phoshorus, no calcium, no magnesium, combinations of these (−P, −Ca; −P, −Mg) or a solution which contained additional potassium but no phosphorus, calcium or magnesium. Ears cultured in the control solution produced grains with higher concentrations of P, Mg, K and Ca than grains from ears which matured on plants in the field. All the −P treatments produced grains with lower concentrations of P and higher concentrations of Mg, K and Ca than the plants grown in the field. The appearance and elemental composition of the globoid crystals in the aleurone cells support the hypotheses that the size of globoid crystals is regulated, in part, by the amount of phosphorus deposited in the grain and also by the relative proportions of K, Mg and Ca in the tissue. The culture of individual ears of cereal plants is a useful technique by which to study the transfer to and deposition of mineral nutrients in cereal grains.

Using flow cytometry, we determined relative DNA amounts expressed as C-values, of large numbers of nuclei isolated from embryo root tips in three seed lots of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum). The nuclei in embryos of fully matured dry pepper and tomato seeds predominantly revealed 2C signals, which indicates that the cells of maturing seeds have arrested at G1 phase of the cell cycle. Priming of pepper and tomato seeds in −1.1, −1.3 or −1.5 MPa polyethylene glycol (PEG) for 14 days reduced the mean time to germination. In all seed lots an induction of 4C signals was found after priming, indicating that during priming the cells of the embryonic root tip had replicated their DNA and arrested at the G2 phase of the cell cycle. For both tomato and pepper, the frequency of 4C signals was highest at the lowest PEG concentration (osmotic potential of −1.1 MPa). A significant inverse correlation was found between the frequency of root tip cells expressing 4C DNA signals and the mean time to germination for each individual pepper and tomato seed lot. However, the effect of a specific osmotic treatment differed between seed lots of the same cultivar, with regard to both the decrease in the mean time to germination and to the percentage of 4C cells, suggesting specific differences among seed lots in the response to priming. The present results indicate that flow cytometry can be used to follow the progress in nuclear replication activities during priming. The advancement in DNA synthesis can thus be used to measure the efficiency of a priming treatment with regard to enhancement of seed lot germination rate.

The cytoplasm of soybean seed axes is known to undergo a glass transition as moisture content decreases. In this study we have investigated whether the glass transition is reflected in the macroscopic mechanical properties of the seeds. Constant-deformation-rate compression tests and constant-deformation relaxation tests were performed with a cylindrical indenter on soybean cotyledons equilibrated to moisture contents ranging from 2% to 16% (dry basis). The apparent modulus of elasticity was found to rise sharply as moisture content declined below 10%, showing an inflection point at 9.8%, and a relatively constant elasticity below 7%. Other measures of mechanical behaviour were also constant in the drier ranges, including linear deformation, and plastic deformation. Each of these viscoelastic characteristics showed an inflection point or a departure from a linear function in the region of 7.5–9.8% moisture content, the hydration region below which the tissue is in a glassy state. It is suggested that the viscoelastic qualities of dry soybeans are principally a consequence of the tissues being in the glassy state.

Relationships have been investigated between gene transcript abundance, enzyme activities and storage product accumulation in developing maize (Zea mays L.) kernels from 10 to 55 days postpollination (DPP). At the early stages of kernel development, there was very little increase in dry weight but kernels accumulated high concentrations of sugars and amino acids. At the end of this ‘lag’ phase (at 15 DPP), many transcripts appeared with little evidence of their translation. The initiation of the kernel-fill period at 20 DPP was characterized by a sudden rise in total RNA, increases in enzyme activities, and the initiation of storage product accumulation. Zein accumulation during this phase was highly correlated with α-zein transcript abundance. Starch accumulation was correlated with both the activity of ADP-GIc pyrophosphorylase and the abundance of gene transcripts encoding this enzyme (Shrunken-2 and Brittle-2). DNA content of kernels increased linearly up to 30 DPP as a result of endoreplication, but had no apparent relationship to gene expression. DNA may accumulate as a storage product. Kernel-fill terminated when the moisture content fell below 36% and was marked by a decline of transcripts and a reduction of enzyme activities.

The effect of accelerated aging (AA) and seed priming on specific seed enzymes was studied in soybeans. Aging seeds for 48 and 96 h at 41°C and 100% humidity reduced germination from 92% to 68% and 0%, respectively. Malate dehydrogenase (MDH) glutamate dehydrogenase (GDH), and esterase were assayed in extracts from dry and imbibing seeds. Malate dehydrogenase activity was the least affected by AA treatments. Certain GDH isozyme activity was reduced by 17% after 48 h of AA and almost completely lost after 96 h. Imbibing for 24 h increased GDH activity in 48 h-accelerated-aged seeds but not the 96 h-aged seeds. Two esterase isozymes in dry seeds showed a 77% loss of activity after 48 h of AA. This treatment increased the total esterase activity, with the greatest activity observed after 96 h of AA. This was due to the appearance of a diffuse high molecular weight (HMW) region of activity observed on native polyacrylamide gels and specifically induced in the embryonic axes. Imbibing 48-h-accelerated-aged seeds for 48 h caused a reappearance of the two esterase enzymes lost during aging. The HMW esterase staining region also appeared after imbibing unaged seeds for 24 h. Seed priming, caused a response similar to imbibition for each enzyme except the HMW esterase activity. Priming 48-h-accelerated-aged seeds reduced the HMW esterase activity. This was not observed during imbibition of aged seeds and indicates the first identification of a specific enzymatic response unique to a seed priming treatment.

Pea seed development on the mother plant consists of three phases, all limited by water concentration (WC). The first (P1) or embryogenesis sensu stricto takes place at constant WC (stable at 80%). During the phase P2, cotyledon filling or maturation, WC decreases linearly from 80 to 55% (physiological desiccation) but the water content stays constant while the dry weight increases until it stops abruptly (at 55% WC), at this time, the seed has almost reached its final dry weight, its maturity mass or physiological maturity. The third phase, P3, consists of a fast desiccation which leads to a WC of 18–14%, where the seed is mature and ready to harvest. Similar events occur in other grain legumes, in cereals where mass maturity is attained at a lower WC (close to 40%) and in other species including crop or weed species. An elementary model of pea seed dry-matter accumulation, based on the constancy of water content (P1) and the linear decrease of WC from 80 to 55% (P2), allows us to define a coefficient α linked to WC and to calculate dry matter changes versus α. This model, taking account of WC in other species, can be generalized easily. Maturation of the somatic embryo, occurring under conditions very close to those present in vivo around the zygotic embryo, follows a pattern of decrease of WC similar to that of the zygotic embryo. We expect that if cell number is similar in the somatic and the zygotic embryo, synseeds will be ready for trade in the near future since control of all the processes that lead to zygotic-like embryoids is now available.

Secondary dormancy (thermodormancy) in Agrostemma githago L. seeds, caused by imbibing the seeds for 4 d at 35°C in the dark, is broken under the influence of cyanide (10 mM), azide (1 mM or BAP (0.44 mM). Both respiration inhibitors (cyanide and azide) and BAP increase the synthesis of five specific soluble proteins in the embryo axes and cotyledons. The activity of β-cyanoalanine synthase (β-CAS, EC 4.4.1.9.), which is able to detoxify cyanide, decreased in response to cyanide and azide. The relevance of these findings to the breaking of thermodormancy is discussed.